Acute exercise preferentially redeploys NK-cells with a highly-differentiated phenotype and augments cytotoxicity against lymphoma and multiple myeloma target cells.

NK-cells undergo a "licensing" process as they develop into fully-functional cells capable of efficiently killing targets. NK-cell differentiation is accompanied by an increased surface expression of inhibitory killer immunoglobulin-like receptor (KIR) molecules, which is positively associated with cytotoxicity against the HLA-deficient K562 cell line. NK-cells are rapidly redeployed between the blood and tissues in response to acute exercise, but it is not known if exercise evokes a preferential trafficking of differentiated NK-cells or impacts NK-cell cytotoxic activity (NKCA) against HLA-expressing target cells. Sixteen healthy cyclists performed three 30-min bouts of cycling exercise at -5%, +5%, and +15% of lactate threshold. Blood samples obtained before, immediately after, and 1h after exercise were used to enumerate NK-cells and their subsets, and determine NKCA and degranulating subsets (CD107+) against cell lines of multiple myeloma (U266 and RPMI-8226), lymphoma (721.221 and 221 AEH), and leukemia (K562) origin by 4 and 10-color flow cytometry, respectively. Exercise evoked a stepwise redeployment of NK-cell subsets in accordance with differentiation status [highly-differentiated (KIR+/NKG2A-) >medium-differentiated (KIR+/NKG2A+)>low-differentiated (KIR-/NKG2A+)] that was consistent across all exercise intensities. NKCA per cell increased ∼1.6-fold against U266 and 221 AEH targets 1h post-exercise and was associated with a decreased proportion of NK-cells expressing the inhibitory receptor CD158b and increased proportion of NK-cells expressing the activating receptor NKG2C, respectively. We conclude that exercise evokes a preferential redeployment of NK-cell subsets with a high differentiation phenotype and augments cytotoxicity against HLA-expressing target cells. Exercise may serve as a simple strategy to enrich the blood compartment of highly cytotoxic NK-cell subsets that can be harvested for clinical use.

Bigley AB ，Rezvani K ，Chew C ，Sekine T ，Pistillo M ，Crucian B ，Bollard CM ，Simpson RJ ... -  《-》

Acute exercise preferentially redeploys NK-cells with a highly-differentiated phenotype and augments cytotoxicity against lymphoma and multiple myeloma target cells. Part II: impact of latent cytomegalovirus infection and catecholamine sensitivity.

We showed previously that acute exercise is associated with a preferential redeployment of highly-differentiated NK-cells and increased cytotoxicity against HLA-expressing tumor cell lines during exercise recovery. In this part II study, we retrospectively analyzed these findings in the context of latent cytomegalovirus (CMV) infection and performed additional experiments to explore potential mechanisms underpinning the marked reduction in NK-cell redeployment with exercise in CMV-seropositive individuals. We show here that latent CMV infection impairs NK-cell mobilization with exercise, only when the intensity of the exercise bout exceeds the individual blood lactate threshold (BLT). This impaired mobilization is associated with increased proportions of poorly exercise-responsive NK-cell subsets (NKG2C+/KIR-, NKG2C+/NKG2A-, and NKG2C+/CD57+) and decreased NK-cell β(2)-adrenergic receptor (AR) expression in those with CMV. As a result, NK-cell production of cyclic AMP (cAMP) in response to in vitro isoproterenol (synthetic β-agonist) stimulation was drastically lower in those with CMV (6.0 vs. 20.3pmol/mL, p<0.001) and correlated highly with the proportion of NKG2C+/CD57+ NK-cells (R(2)=0.97). Moreover, NK-cell cytotoxic activity (NKCA) against the K562 (36.6% vs. 22.7%, p<0.05), U266 (23.6% vs. 15.9%, p<0.05), and 221.AEH (41.3% vs. 13.3%, p<0.001) cell lines was increased at baseline in those infected with CMV; however, latent CMV infection abated the post-exercise increase in NKCA as a result of decreased NK-cell mobilization. Additionally, NKCA per cell against the U266 (0.24 vs. 0.12, p<0.01), RPMI-8226 (0.17 vs. 0.11, p<0.05), and 221.AEH (0.18 vs. 0.11, p<0.05) cell lines was increased 1h post-exercise (relative to baseline) in CMV-seronegative subjects, but not in those infected with CMV. Collectively, these data indicate that latent CMV infection may compromise NK-cell mediated immunosurveillance after acute exercise due to an increased proportion of "CMV-specific" NK-cell subsets with impaired β-adrenergic receptor signaling pathways.

Bigley AB ，Rezvani K ，Pistillo M ，Reed J ，Agha N ，Kunz H ，O'Connor DP ，Sekine T ，Bollard CM ，Simpson RJ ... -  《-》

Latent cytomegalovirus infection enhances anti-tumour cytotoxicity through accumulation of NKG2C+ NK cells in healthy humans.

Cytomegalovirus (CMV) infection markedly expands NKG2C+/NKG2A- NK cells, which are potent killers of infected cells expressing human leucocyte antigen (HLA)-E. As HLA-E is also over-expressed in several haematological malignancies and CMV has been linked to a reduced risk of leukaemic relapse, we determined the impact of latent CMV infection on NK cell cytotoxicity against four tumour target cell lines with varying levels of HLA-E expression. NK cell cytotoxicity against K562 (leukaemia origin) and U266 (multiple myeloma origin) target cells was strikingly greater in healthy CMV-seropositive donors than seronegative donors and was associated strongly with target cell HLA-E and NK cell NKG2C expression. NK cell cytotoxicity against HLA-E transfected lymphoma target cells (221.AEH) was ∼threefold higher with CMV, while NK cell cytotoxicity against non-transfected 721.221 cells was identical between the CMV groups. NK cell degranulation (CD107a(+) ) and interferon (IFN)-γ production to 221.AEH cells was localized almost exclusively to the NKG2C subset, and antibody blocking of NKG2C completely eliminated the effect of CMV on NK cell cytotoxicity against 221.AEH cells. Moreover, 221.AEH feeder cells and interleukin (IL)-15 were found to expand NKG2C(+) /NKG2A(-) NK cells preferentially from CMV-seronegative donors and increase NK cell cytotoxicity against HLA-E(+) tumour cell lines. We conclude that latent CMV infection enhances NK cell cytotoxicity through accumulation of NKG2C(+) NK cells, which may be beneficial in preventing the initiation and progression of haematological malignancies characterized by high HLA-E expression.

Bigley AB ，Rezvani K ，Shah N ，Sekine T ，Balneger N ，Pistillo M ，Agha N ，Kunz H ，O'Connor DP ，Bollard CM ，Simpson RJ ... -  《-》

Autologous serum collected 1 h post-exercise enhances natural killer cell cytotoxicity.

Natural Killer cells are cytotoxic lymphocytes that recognize and eliminate tumor cells. Exercise enhances NK cell cytotoxic activity (NKCA), yet the underlying mechanisms are not fully understood. Exercise-induced shifts in NK-cell subsets has been proposed as one mechanism. Alternatively, exercise alters stress hormone and cytokine levels, which are also known to affect NKCA. Determine the role(s) of exercise-induced shifts in the proportions of NK-cell subsets found in the blood, and changes in serum IL-2, IL-6, IL-12, IFN-γ, TNF-α and cortisol, on exercise-induced changes in NKCA. Twelve adults cycled 30 min at 115% of their lactate threshold power. Peripheral blood mononuclear cells (PBMCs) and serum were isolated from blood collected pre-, post-, and 1 h post-exercise. To investigate the effect of shifts in NK-cell subsets, pre-, post- and 1 h post-exercise NK cells were incubated with target cells (K562 and U266) in the presence of autologous pre-exercise serum. The effects of hormones and cytokines released during exercise were determined by incubating pre-exercise PBMCs with tumor target cells (K562 and U266) in the presence of pre-, post-, and 1 h post-exercise serum. NKCA and phenotypes were assessed by flow cytometry. Although exercise mobilized high-differentiated NK cell subsets (NKG2A-/KIR+), NKCA per cell was not altered post-exercise in the presence of pre-exercise serum. Conversely, 1 h post-exercise serum significantly increased the cytotoxicity of pre-exercise NK cells against HLA-expressing target cells (U266). This increase associated with lower levels of cortisol, and occurred when serum contained higher levels of IFN-γ. Exercise-induced shifts in NK-cell subsets did not fully explain changes in NKCA. Rather, factors present in serum during exercise recovery enhanced NKCA against target cells. Our results suggest lower cortisol and higher IFN-γ levels may explain exercise-induced changes in NKCA.

Gupta P ，Bigley AB ，Markofski M ，Laughlin M ，LaVoy EC ... -  《-》

NKG2A Expression Is Not per se Detrimental for the Anti-Multiple Myeloma Activity of Activated Natural Killer Cells in an In Vitro System Mimicking the Tumor Microenvironment.

Mahaweni NM ，Ehlers FAI ，Sarkar S ，Janssen JWH ，Tilanus MGJ ，Bos GMJ ，Wieten L ... -  《Frontiers in Immunology》